Paclitaxel is a useful therapeutic agent for various cancers including, but not limited to, ovarian cancer, breast cancer, and lung cancer. This taxane-type diterpene was first identified after isolation from Taxus brevifolia NUTT, a plant belonging to the genus Taxus, family Taxaceae. While paclitaxel can be found in all parts of the plant body, the bark has been found to have the highest concentration. At present, paclitaxel is collected from a natural or cultivated plant body. However, plants belonging to the genus Taxus grow very slowly, taking more than 10 years to grow to a height of 20 cm above the ground. Further, stripping of the bark generally results in tree death. Thus, isolating from natural sources the large amounts of paclitaxel needed for therapeutic uses is extremely difficult, if not impossible.
Attempts have been made to chemically synthesize paclitaxel. However, paclitaxel is a large, structurally complex molecule and large scale synthesis from simple, available chemicals is currently not a viable commercial alternative.
Semi-synthetic production via chemical attachment of a side chain to the agriculturally produced paclitaxel precursor, 10-deacetyl baccatin, has also been proposed (U.S. Pat. No. 4,924,011; U.S. Pat. No. 5,015,744). 10-Deacetyl baccatin is obtained from the needles of trees of the Taxus species. However, obtaining this precursor is by no means trivial and 10-deacetyl baccatin content of the needles may not be as high as initially reported. Thus, semi-synthetic production is expensive and also unlikely to be able to supply the necessary amount of paclitaxel required for its chemotherapeutic applications.
The most promising means for producing sufficient amounts of paclitaxel necessary for therapeutic applications is through plant cell cultures.
A variety of methods for producing paclitaxel and other taxanes or taxane-related compounds via cultured plant cells have been described.
U.S. Pat. No. 5,019,504 describes a method for producing paclitaxel and derivatives thereof via cultured cells of Taxus brevifolia. However, the yield from this method ranges between 1 to 3 mg/L and is insufficient for industrial applications.
U.S. Pat. No. 5,015,744 describes a method for production of baccatin III via plant cell culture, which can then be used in semi-synthetic production of paclitaxel.
U.S. Pat. No. 5,407,816 and WO 93/17121 describe a method for producing paclitaxel and paclitaxel-like compounds from Taxus cells inoculated on a nutrient medium. In this method, paclitaxel is produced in an amount of at least 10-fold greater than that produced by native Taxus cells. Using this method, Taxus chinensis cells produced a yield of 153 mg/L of paclitaxel and taxanes. However, the requirements of the nutrient media are quite complicated and growth conditions are quite limited, thus rendering industrial applicability of this method also questionable.
Methods for increasing paclitaxel production via addition of stimulators to plant cell culture medium have also been proposed. For example, U.S. Pat. No. 5,637,484 describes a method for increasing paclitaxel production by addition of jasmonate and Ag-containing compounds to the culture medium. Addition of methyl jasmonate to increase production of paclitaxel in Taxus cell cultures was also disclosed by Yukimune et al. (Nature Biotechnology 1996 14:1129-1132) and Mirjalili and Linden (Biotechnol. Prog. 1996 12:110-118). WO 97/44476 describes methods for producing paclitaxel, baccatin III and other paclitaxel-like compounds in high yield from Taxus cells cultured with enhancement agents such as silver ion or complex, jasmonic acid, auxin-related growth regulators, and inhibitors of the phenylpropanoid pathway such as 3,4-methylenedioxy-6-nitrocinnamin acid. U.S. Pat. No. 5,871,979 describes a method for producing paclitaxel in high yields from semi-continuous cultures of Taxus genus plant wherein the plant cells are inoculated on a medium containing sugar. U.S. Pat. No. 6,248,572 also describes methods for producing paclitaxel in large amounts by culturing Taxus genus plant cells in a culture medium containing sugar alone or sugar in combination with AgNO3. EP 0 727 492 A2 describes a method for producing taxane-type diterpenes in a plant cell or tissue by culturing the cell or tissue in the presence of a coronatine, a bacterium which produces a coronatine, a culture solution or a culture extract of such bacterium, cyclic polysaccharides, fatty acids or an imino or amino derivative of jasmonic acid. Furmanowa et al. (Biotechnology Letters 2000 22:1449-1452) describe methods for increasing paclitaxel production in Taxus cuspidate cell culture via addition of vanadyl sulfate, phenylalanine, or chitosan and methods for increasing baccatin III production in Taxus media cell culture via addition of aminobenzoic acid. Additionally, studies performed to reveal compounds that may enhance paclitaxel production in vivo suggest that β-phenylalanine may increase paclitaxel production; however, in the production study only a mixture of α and β-phenylalanine is fed. β-phenylalanine alone was not used (WO 97/44476).